The adult stem cell is a fundamentally important cell type that lies at the center of regenerative medicine and disease pathology. Its existence is essential for life-long growth, maintenance of somatic tissues, and repair of damaged cells. Conversely, the misregulation of adult stem cells may lie at the heart of diseases such as cancer. From this perspective, a thorough understanding of their biological properties and the mechanisms that govern their unique cellular capabilities is required. The melanocyte, a pigment cell, and its adult stem cell present an opportunity to ask fundamental questions about the origin and development of this unique class of cell. The zebrafish offers a highly tractable model system for studying the melanocyte stem cell, or MSC. Two types of melanocytes exist, those that develop directly from the neural crest in early development and those that are derived later from MSCs. In aim 1, clonal analysis will be used to determine the relationship between the embryonic direct- developing melanocyte lineage and the stem cell-derived melanocyte lineage. Clones will be induced using X-rays at various developmental stages to determine when the two lineages diverge, giving insight into how early in development an embryo purposefully sets asides cells for future maintenance of somatic lineages. A common property of stem cells is the ability to generate multiple closely related differentiated cell types. Clonal analysis of adult fins led to the hypothesis that a single adult stem cell may give rise to two types of pigment cells in zebrafish, melanocytes and xanthophores. By using a transgenic line of zebrafish (MAZe) developed for inducing clones with strict temporal control, the notion of whether adult stem cells in the fin are multipotent or unipotent will be tested in aim 2. In addition, the mechanism by which a multipotent stem cell generates two differentiated cell types will be investigated. To thoroughly explore the mechanisms that govern adult stem cell biology, it is necessary to develop a transgenic line that marks the MSC in zebrafish (aim3). Three lines recently developed in the lab will be challenged with a panel of drugs that have been shown to affect the MSC. Combining a MSC marker with drugs that have definitive molecular targets provides an opportunity to understand the genes and pathways responsible for stem cell identity and the process by which a stem cell chooses to divide and generate daughters for differentiation. PUBLIC HEALTH RELEVANCE: Adult stem cells play a critical biological role in the life of an organism, able to rapidly produce new cells during growth and regenerate cells when natural processes and unexpected injury lead to tissue loss. This research proposal uses the zebrafish melanocyte, a type of pigment cell, to ask when the adult melanocyte stem cell originates in the development of an animal, if and how an adult stem cell generates multiple differentiated cell types, and aims to identify a melanocyte stem cell marker that will allow the visualization and further study of this unique cell type. The development of a critical tool to study the melanocyte stem cell opens the door to a more fundamental understanding of what defines an adult stem cell, and could ultimately provide insights into mechanisms that play a role in melanoma and other pigment disorders.